mmg_233_2014_genetics_genomicsfandomcom-20200215-history
Blue and White Screening
Overview and History Blue and White screening is a phenotypic screening method used in molecular cloning experiments. "Blue and White" refers to colors seen on the plate that distinguish the transformed colonies versus the colonies that do not have vector (Figure 1). "Screening" refers to that the scientist can quickly see or screen the plate to see what colonies are blue and which ones are white indicating the presence of the vector. (Add History) Review of Molecular Cloning and Use Molecular Cloning is widely used in microbiology and molecular genetics labs. Molecular Cloning is used to amplify a specific gene product and transform it into a different species. This overall process is laid out in Figure 2. 1. PCR - Polymerase Chain Reaction: '''Polymerase Chain Reaction amplifies a specific region in the DNA sequence. The polymerase can only amplify a certain region because it is denoted with forward and reverse primers running in both the 5' to 3' and 3' to 5' directions. PCR works by exponentially amplifying the DNA. Therefore after about thirty cycles, there are many hundreds of thousands of copies of that particular region. 2. '''Cloning the PCR product (insert) into the plasmid (vector): Now that you have multiple copies of your PCR product you need to clone it into your plasmid. There are many different plasmids to choose from. The plasmid you choose must have a selectable marker like antibiotic resistance, a reporter gene such as LacZ, and a multiple cloning site (MCS). The plasmid (Figure 3) used most commonly is pBluescript II SK (or pGEM). The insert does not just add anywhere in the plasmid. It is added to the MCS or multiple cloning site that is right in the middle of the LacZ gene. 3. 'Transform plasmid with insert into component cells: '''The next step in this process is to transform or put the plasmid with the PCR product in it into a component cell. "Component" refers to the cell's ability to take up extra cellular DNA. ''E. coli cells are usually used as component cells because they grow very fast and are able to recover from the transformation. Transformation can done in two different ways. One way is to electroporate the cells. This makes use of a electric current to make holes in the membrane of the cell through which the plasmid can enter. Another method is to make the cells chemically competent. This uses changes in temperature to open the "pores" on the membrane of the competent cells and have the plasmid enter there. 4.'''Recovery Step and Blue-White screening: '''After the transformation step, the cells must be able to recover and be allowed to repair themselves. This usually requires that the cells incubate overnight and then are plated the next day. As seen in Figure 2, five different plates are set up in a blue-white screening process- a positive control, a negative control, and different amounts of the transformed cells. These are all plated on LB Amp plates and covered with X-gal. Mechanism behind Blue-white screening (Alpha Complementation) Blue-white screening is mainly based on the reporter gene LacZ. LacZ is a gene that is able to produce a protein called β-galactosidase. To produce a functional β-galactosidase enzyme, it needs the alpha and beta module. The LacZ gene in the plasmid has the alpha module of the enzyme and the chromosomal DNA carries the other (beta) module. This whole enzyme is able to break down a form of lactose (i.e. X-gal). Since the MCS is right in the middle of the LacZ gene, if an insert is added it will disrupt the gene and the alpha module of this enzyme will not be produced. On the other hand if the insert is not present, both the alpha and beta modules will be produced therefore creating a functional enzyme. This detection of the insert is seen through the phenotype of the colonies. When X-gal is broken down by β-galactosidase, it appears to have a blue color. This is because when β-galactosidase breaks down X-gal, its product 5-bromo-4-chloro-indoxyl dimerizes and oxides to form 5,5'-dibromo-4,4'-dichloro-indigo (an insoluble blue pigment). When the insert is present, the enzyme is not formed and the colony looks white to the human eye. References Blue White Screening, Wikipedia, retrieved: 9/25/14 Alpha Complementation, from UTEP.edu, retreived: 9/25/14. "Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the β-galactosidase structural gene of Escherichia coli", Agnes Ullmann, François Jacob, Jacques Monod, Journal of Molecular Biology Volume 24, Issue 2, 14 March 1967, Pages 339–343.